This program project consisting of three projects and three cores aims at elucidating molecular mechanisms that are responsible for membrane fusion in presynaptic exocytosis. The molecular machine at the center of this process is the SNARE complex that assembles from its components during presynaptic membrane fusion. Energy derived from SNARE complex folding and assembly drives the kinetically blocked fusion of vesicle and cell membrane bilayers. Regulatory proteins including synaptotagmin, complexin, and Munc18 render SNARE complex formation and fusion sensitive to calcium and control SNARE assembly and fusion by mechanisms that are still poorly understood at the structural mechanistic level. A group of talented biochemists, structural biologists, biophysicists, and cell biologists has been assembled in this program project to jointly tackle several key questions of presynaptic membrane fusion. SNARE assembly and fusion intermediates will be structurally and functionally characterized and correlated;the structural and biophysical basis of the regulation of SNARE assembly by accessory proteins and its coupling to fusion will be elucidated;and the evolution of the supramolecular architecture of nascent and mature fusion pores will be examined. The approaches range from high-resolution molecular spectroscopy and structural biology to biochemical and molecular and biological dissections of the process with judiciously selected mutants, to advanced optical and cell biological methods. PUBLIC HEALTH RELEVANCE: Fusion of synaptic vesicles with the cell membranes of neurons is a key element of synaptic transmission. Defects in synaptic transmission lead to epilepsy, depression, and other neurological disorders. Understanding the molecular mechanism and structural basis of membrane fusion at the synapse will ultimately help to combat these diseases.